Apoptosis is an orderly process of cellular elimination, which ensures complete disassembly and disappearance of damaged cells. Recently the concept of apoptotic cell elimination was expanded and programmed cell death is no longer viewed as an individual cellular event. The complete apoptotic process now includes two phases: the self-driven cell disassembly and externally-controlled elimination of apoptotic cell corpses by phagocytizing cells. The second phagocytic phase is essential, highly conserved and is considered to be even more important than the internal phase of cell disassembly. This is because it ensures the complete degradation of the dying cell's DNA, preventing release of pathological, viral and tumor DNA and self-immunization. In different cells and species from mammals to flies, only two separate and conserved groups of enzymes are responsible for elimination of cellular DNA in the first and second phases of apoptosis. The nucleases producing DNase I-type DNA cleavage are essential for the first phase of apoptotic execution, whereas a single enzyme DNase II plays a fundamental role in the 'mopping up'phase of apoptosis. The probes which could detect activity of both enzymatic groups would permit comprehensive labeling of apoptotic execution. In this project we will introduce such an approach. Our technology capitalizes on the fact that both types of nucleases produce identifiable signature DNA breaks. The new assay will simultaneously detect both the intracellular and phagocytic phases of apoptotic DNA degradation in tissue sections. It is based on multi-labeling of signature DNA cuts produced by the executioner and phagocytic nucleases. The Specific Aims of the proposal are: 1) To develop a dual-detection apoptotic assay for labeling of internal and phagocytic stages of apoptotic cell disassembly;2) To test the new technology in the model of permanent focal brain ischemia. The new assay will be a unique and practical tool in the apoptosis detection arsenal. It will have broad applicability in biomedicine. Its application to brain ischemia will provide information useful for the development of effective therapeutic interventions. PUBLIC HEALTH RELEVANCE: The proposed project will result in the development of a new assay for the needs of medical diagnostics and pathology. The technology will allow precise evaluation of the effects of therapy in diseases where cell death and DNA damage have prognostic value, such as stroke, Alzheimer's disease, and various cancers.